Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Maspin, a novel mammary serine protease inhibitor, was shown to have tumor suppressing activity (Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R. (1994) Science 263, 526-529). In this paper, we report the production of recombinant glutathione S-transferase-maspin fusion protein, expressed in the bacterium Escherichia coli, and recombinant maspin, expressed in the insect Spodoptera frugiperda cells. The fusion protein was purified by glutathione affinity chromatography. Maspin expressed in insect cells was purified by a combination of Bio-Rad AG1-2X anion exchange chromatography and heparin affinity chromatography. The recombinant maspin from insect cells was cleaved at the putative reactive center, as confirmed by protein sequencing. Both recombinant proteins demonstrated strong inhibitory effects on the invasion by two breast tumor cell lines across reconstituted basement membranes and such inhibitory effect was abolished in the presence of the polyclonal antibody made against the reactive center region of maspin. The trypsin-cleaved recombinant maspin did not inhibit invasion, indicating that the inhibitory activity requires the intact putative reactive center. This paper provides evidence that recombinant maspin protein itself inhibits invasion, and supports the role of maspin as a tumor suppressor.
...
PMID:Production, purification, and characterization of recombinant maspin proteins. 798 35

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.
...
PMID:Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor. 811 69

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
...
PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

The 2.4 A crystal structure (R = 0.180) of the serine protease inhibitor ecotin was determined in a complex with trypsin. Ecotin's dimer structure provides a second discrete and distal binding site for trypsin and, as shown by modelling experiments, other serine proteases. The second site is approximately 45 A from the reactive/active site of the complex and features 13 hydrogen bonds, including six that involve carbonyl oxygen atoms and four bridged by water molecules. Contacts ecotin makes with trypsin's active site are similar to, though more extensive than, those found between trypsin and basic pancreatic trypsin inhibitor. The side chain of ecotin Met84 is found in the substrate binding pocket of trypsin where it makes few contacts, but also does not disrupt the solvent structure or cause misalignment of the scissile bond. This first case of protein dimerization being used to augment binding energy and allow chelation of a target protein provides a new model for protein-protein interactions and for protease inhibition.
...
PMID:Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex. 815 87

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.
...
PMID:Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor. 822 33

X protein of hepatitis B virus (HBV) transactivates transcription of various viral and cellular genes. It has been suggested that X protein plays a major role in hepatocarcinogenesis by HBV. The protein possesses amino acid sequence homology to the functionally essential domain of Kunitz-type serine protease inhibitors. This Kunitz domain-like sequence in X protein is indispensable for the transactivation function. To clarify whether X protein has a serine protease inhibitor activity, a search was made for serine proteases which interact with, but not degrade X protein. Tryptase TL2, one of serine proteases in hepatic cells, was found to directly interact with X protein without degradation. Moreover, the activities of tryptase TL2 and an analogous protease were substantially inhibited by X protein. These results suggest that transactivation function of X protein is exerted by modulation of the hepatic serine protease activity, giving rise to quantitative or qualitative change of cellular transcription factor(s) through protection from proteolytic degradation and/or suppression of processing.
...
PMID:Interaction of hepatitis B virus X protein with a serine protease, tryptase TL2 as an inhibitor. 829 Feb 48

The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60D as part of an "aromatic stacking chain." Biochemical inhibition data (Ki values at 37 degrees C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (Ki = 0.18 microM) but a potent inhibitor of trypsin (Ki = 0.023 microM) and streptokinase (Ki = 0.035 microM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.
...
PMID:Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A. 836 61

Equine neutrophil antimicrobial peptide 2 (eNAP-2), a recently described antimicrobial peptide isolated from equine neutrophils, was found to selectively inactivate microbial serine proteases (subtilisin A and proteinase K) without inhibiting mammalian serine proteases (human neutrophil elastase, human cathepsin G, and bovine pancreatic trypsin). Although the primary structure of eNAP-2 resembled that of several known antiproteases that belong to the 4-disulfide core peptide family, this pattern of selectivity is unique. eNAP-2 formed a noncovalent complex with native subtilisin A or proteinase K but did not associate with these enzymes if they had been treated with phenylmethylsulfonyl fluoride, a serine protease inhibitor. The eNAP-2-microbial protease complex was disrupted by boiling or by exposure to low pH. We suggest that eNAP-2 exerted selective antiproteinase activity by binding tightly but noncovalently to the active site of subtilisin A or proteinase K. Since microbial exoproteases may act as virulence factors, the combined antimicrobial and antiprotease activities of eNAP-2 could allow it to play an important role in neutrophil-mediated antimicrobial defenses.
...
PMID:Selective inhibition of microbial serine proteases by eNAP-2, an antimicrobial peptide from equine neutrophils. 851 5

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.
...
PMID:Neutrophil apoptosis induced by proteolytic enzymes. 860 Mar 21

Gelatinolytic metalloproteinases implicated in connective tissue remodeling and tumor invasion are secreted from several types of cells in the form of inactive zymogens. In this report, characterization of gelatinase activity secreted by the BR line of dog mastocytoma cells reveals a phorbol-inducible, approximately 92-kD, Ca2+ - and Zn2+ -dependent proenzyme cleaved over time to smaller, active forms. Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products. The NH2-terminal 13 amino acids of the purified mastocytoma progelatinase are 50-67% identical to those of human, mouse, and rabbit 92-kD progelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore A23187 greatly accelerated proenzyme cleavage, suggesting that a serine protease present in secretory granules hydrolyzed the progelatinase to active fragments. To identify the activating protease, cells were coincubated with ionophore and a panel of selective serine protease inhibitors. Soybean trypsin inhibitor and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mast cell chymase, prevented progelatinase activation. Inhibitors of tryptase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-amidino-2-benzimidazolyl) methane (BABIM), did not. In further experiments using highly purified enzymes, mastocytoma cell chymase activated 92-kD progelatinase in the absence of other enzymes or cofactors; tryptase and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinase B that is directly activated outside of the cell by exocytosed chymase, and provide the first demonstration of a cell that activates a matrix metalloproteinase it secretes by cosecreting an activating enzyme. In mastocytomas, this pathway may facilitate tumor invasion of surrounding tissues, and in normal mast cells, it could play a role in tissue remodeling and repair.
...
PMID:Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast cell chymase. 860 22


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---